Journal of Chromatography B (v.830, #1)

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of scutellarin in human plasma has been developed. Samples were prepared using solid phase extraction and analyzed on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol–water (0.1% formic acid), using gradient procedure. The analyte and internal standard baicalin were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.2–20.0 ng/mL. The lower limit of quantification (LLOQ) was 0.2 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.4%. The accuracy determined at three concentrations (1.0, 5.0 and 10.0 ng/mL for scutellarin) was within ±5.0% in terms of relative error. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of scutellarin guttate pills in 20 healthy volunteers.
Keywords: Scutellarin; LC–MS/MS; Pharmacokinetics;

Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS by Tom Delahunty; Lane Bushman; Courtney V. Fletcher (6-12).
An LC/MS/MS assay for the determination of tenofovir (TNF) was developed and validated for use with the EDTA anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was used to produce a protein-free extract. Chromatographic separation was achieved with a Polar-RP Synergi, 2.0 mm × 150 mm, reversed-phase analytical column. The mobile phase was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable limit of 10 ng/ml when 250 μl aliquots were analyzed. The usefulness of this LC/MS/MS method to routinely monitor plasma concentrations of TNF was demonstrated along with its ability to assist in the performance of pharmacokinetic studies.
Keywords: Tenofovir (TNF); Adefovir (PMEA); LC/MS/MS; Plasma;

MS23 is a vasodilator with unique dual action pharmacological profile to inhibit type 4 PDE and antagonize L-type calcium channels. We validated an analytical protocol for MS23 in rat plasma using high performance liquid chromatography (HPLC). A C18 column and a phosphate/acetonitrite buffer were used for chromatographic separation. UV detection was performed at 307 nm. The calibration curve for MS23 was linear in the range from 50 to 10,000 ng/ml. The limit of quantification (LOQ) was 50 ng/ml. The results demonstrate that the method has linearity (R  = 0.9989), specificity, and acceptable precision/accuracy. This method is simple, economic, and sufficient for in vivo pharmacokinetic studies on the compound.
Keywords: Validation; MS23; Antihypertensive; PDE4 inhibitor; Dual action; Calcium channel antagonist;

SPE–GC–MS for the sampling and determination of unmetabolized styrene in urine by C. Prado; P. Marín; P. Simon; J.F. Periago (18-24).
The urinary excretion of unmetabolized styrene can be a very good indicator for biomonitoring styrene in occupationally exposed people. The use of a new urine sampling system, involving a solid-phase extraction cartridge, offers several advantages for determining styrene. The advantages are especially related to the pre-analytical phase of styrene determination, which may be influenced by many variables. The effect on styrene recovery of sorbent type, eluting solvent, elution volume, elution flow-rate, and the addition of methanol to the washing solvent, was evaluated by experimental design methodology. As a result, Oasis HLB cartridges were selected for urine sampling, as well as 1.5 mL of ethyl acetate at 0.5 mL/min for eluting the retained styrene. These conditions were then applied to the validation of the solid-phase extraction combined with GC–MS method for the sampling and analysis of unmetabolized styrene in urine. The overall uncertainty was in the 12–22% range and the limit of detection was 2.2 μg/L for a 4 mL urine sample. The stability of styrene has been studied both in cartridges and in vials under different storage periods. After 1 month period the styrene stored on cartridges at room temperature remained stable, whereas this is not the case for styrene recovery from vials. The results obtained indicate that on-site solid-phase extraction of urine can provide a simple, accurate and reproducible sampling and analytical method for the biomonitoring of styrene in urine.
Keywords: Styrene; Urine; SPE; GC–MS; Biomonitoring;

Ultra-sensitive determination of Formoterol in human serum by high performance liquid chromatography and electrospray tandem mass spectrometry by Daniel Gerhard Mascher; Karl Zech; Rüdiger Nave; Klaus Michael Kubesch; Hermann Josef Mascher (25-34).
An analytical method was developed and validated to determine Formoterol in human serum in the range from 0.40 to 100.24 pg/mL by high performance liquid chromatography and tandem mass spectrometry (HPLC–MS/MS) due to the lack of efficient methods to determine very low levels of Formoterol in serum and plasma. Serum was diluted by water and mixed with the internal standard (d6-Formoterol). Formoterol and internal standard were extracted using a cation-exchange solid phase column (SCX-3). After eliminating endogenous serum constituents through washing steps with water and methanol, elution took place using methanol/ammonia. After evaporation of the elution liquid the residue was redissolved and analyzed by HPLC–MS/MS with electrospray ionisation (ESI) in positive mode. A gradient between 10 mM ammonium formate and acetonitrile was used. The inter-batch precision of the calibration standards ranged from 1.55% to 9.01%. The inter-batch accuracy of the calibration standards ranged from 93.37% to 107.30%. The lower limit of quantitation (LLOQ, 0.40 pg/mL) had a precision of 19.67% and an accuracy of 96.78%. Comparable results were obtained for quality control samples. Stability in human serum was given over three freeze/thaw cycles and 2 h at room temperature. Formoterol in human serum was stable for at least 6 months below −20 °C. This method has been used widely for quantifying Formoterol after inhalation of 9–36 μg of the drug by volunteers. A cross validation with human plasma versus serum was performed after this method was successfully validated in human serum.
Keywords: Formoterol; Human serum; Ultra sensitive method; Tandem mass spectrometry; Solid phase extraction; Liquid chromatography;

Determination of the heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin in plasma by liquid chromatography–electrospray mass spectrometry by Kyunghwa Hwang; Charity D. Scripture; Martin Gutierrez; Shivaani Kummar; William D. Figg; Alex Sparreboom (35-40).
A rapid method was developed for the quantitative determination of the novel heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), in human plasma. Calibration curves were constructed, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step extraction with ethyl acetate of 0.5-ml samples. The analysis was performed in the range of 1–100 ng/ml on a column (75 mm × 2.1 mm internal diameter with 3.5 μm C18 particle size), using 55% methanol in water containing formic acid as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always <8% and <10% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of 17-DMAG in a cancer patient.
Keywords: 17-DMAG; LC/MS; Plasma; Pharmacokinetics; Cancer;

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method by M. Romeu-Nadal; S. Morera-Pons; A.I. Castellote; M.C. López-Sabater (41-46).
Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.
Keywords: Human milk; Ascorbic acid; Vitamin C; HPLC; Enzymatic method;

Analysis of microperoxidases using liquid chromatography, post-column substrate conversion and fluorescence detection by Rob Haselberg; Christel Hempen; Suze M. van Leeuwen; Martin Vogel; Uwe Karst (47-53).
A liquid chromatographic method with on-line activity determination for microperoxidases has been developed. After enzymatic digestion of a cytochrome, possibly under formation of microperoxidases, the product mixture is separated by reversed-phase liquid chromatography. The products first pass a diode-array detector, and are then subjected to a reaction with 4-(N-methylhydrazino)-7-nitro-2,1,3-benzooxadiazole (MNBDH) and hydrogen peroxide. In a reaction coil, microperoxidases catalyze the reaction under formation of the fluorescent 4-(N-methylamino)-7-nitro-2,1,3-benzooxadiazole (MNBDA). Quantification of the microperoxidases is performed using a fluorescence detector at an excitation wavelength of 470 nm and an emission wavelength of 545 nm, respectively. For this LC-based detection system, limits of detection are 3 × 10−8  mol/L, limits of quantification are 9 × 10−8  mol/L, and a linear range from 9 × 10−8  mol/L to 3 × 10−6  mol/L is obtained for the microperoxidases MP-9 and MP-11. A highly active microperoxidase MP-6 was found in the reaction of cytochrome c from bovine heart with protease from streptomyces griseus.
Keywords: Microperoxidases; HPLC; Post-column derivatization; Fluorescence detection; MS detection; Digestion;

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M 1 ), 5-pregnene--ol-20-one (M 2 ), 5-pregnene-, 20-diol (M 3 ), 5-pregnene--ol-16, 17-epoxi-20-one (M 4 ) and 5,16-pregnadien-, 11-diol-20-one (M 5 ) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 μm, 100 mm × 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS–MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56–400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M3 and M5 where linearity was over the 3.13–400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at −60 °C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.
Keywords: LC-MS–MS; Hypolipidaemic agent; 16-Dehydropregnenolone; Rabbit plasma; MRM;

Direct determination of glucuronide and sulfate of p-hydroxymethamphetamine in methamphetamine users’ urine by Noriaki Shima; Hiroe Tsutsumi; Tooru Kamata; Mayumi Nishikawa; Munehiro Katagi; Akihiro Miki; Hitoshi Tsuchihashi (64-70).
Two conjugates of p-hydroxymethamphetamine (p-OHMA), p-OHMA-glucuronide (p-OHMA-Glu) and p-OHMA-sulfate (p-OHMA-Sul) have been identified in methamphetamine (MA) users’ urine by using liquid chromatography-high resolution tandem mass spectrometry (LC–HRMS-MS). The synthesis of p-OHMA-Glu and p-OHMA-Sul, and an LC–MS procedure for the simultaneous determination of MA and its four metabolites, amphetamine (AP), p-OHMA, p-OHMA-Glu and p-OHMA-Sul, in urine have also been established. After deproteinizing urine samples with methanol, LC–MS employing a C18 semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Based on the established method, p-OHMA-Sul was detected at higher concentrations than p-OHMA-Glu in all of the three urine samples tested. These data suggest that sulfation is a major pathway in the MA phase II metabolism.
Keywords: Methamphetamine; p-Hydroxymethamphetamine; Sulfate; Glucuronide; Urine;

Two novel metabolites of benproperine (BPP), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3-OH-BPP) and 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol (4-OH-BPP), were confirmed by comparison of retention times and mass spectra with those of synthetic standards using liquid chromatography–tandem mass spectrometry. Selective and sensitive procedures were developed for the simultaneous determination of BPP, 3-OH-BPP and 4-OH-BPP in human plasma and urine. The analytes were extracted from plasma sample and enzymatically hydrolyzed urine samples by liquid–liquid extraction, separated through a Diamonsil C18 column (150 mm × 4.6 mm i.d.) and determined by tandem mass spectrometry with an electrospray ionization interface in selected reaction monitoring mode. Dextromethorphan was used as internal standard. The mobile phase consisted of acetonitrile–water–formic acid (34:66:1, v/v/v), and flow-rate was 0.5 ml min−1. This method has a lower limit of quantification (LLOQ) of 60, 4.0 and 4.0 nmol l−1for BPP, 3-OH-BPP and 4-OH-BPP in plasma, 4.9, 4.7 and 2.4 nmol l−1 in urine, respectively. The intra- and inter-run precision were measured to be below 9.2%, and the accuracy was within ±4.3% for the analytes. The method was successfully used to determine BPP, 3-OH-BPP and 4-OH-BPP in plasma and urine for pharmacokinetic investigation. The results indicated residue of 3-OH-BPP in the body at least 192 h after an oral dose of BPP.
Keywords: Benproperine; 3-Hydroxybenproperine; 4-Hydroxybenproperine;

A simple and sensitive liquid chromatography–mass spectrometry method is described for the determination of nicardipine in human plasma. Chromatographic separation of the analyte was achieved on a C18 column using a mobile phase of methanol, water and formic acid (320:180:0.4, v/v/v). Selected ion monitoring (SIM) in positive mode was used for analyte quantification at m/z 480.2 for nicardipine and m/z 256.4 for diphenhydramine. The run time was less than 5 min. The linearity over the concentration range of 0.05–20.0 ng/ml for nicardipine was obtained and the lower limit of quantification was 0.05 ng/ml. For each level of QC samples, inter-day and intra-day precisions (R.S.D.) were ≤9.3 and 11.1%, respectively, and accuracy (RE) was ±4.9%. The present LC–MS method was successfully applied in the pharmacokinetic studies of nicardipine hydrochloride delayed-release tablets in two formulations after oral administration to healthy volunteers.
Keywords: Nicardipine; Liquid chromatography–mass spectrometry; Human plasma;

Quantification of tipranavir in human plasma by high-performance liquid chromatography with UV detection by Emmanuelle Giraud; Elisabeth Rey; Jean-Marc Tréluyer; Gérard Pons; Vincent Jullien (86-90).
A simple method for the quantification of tipranavir, a new non-peptidic protease-inhibitor, was developed. An internal standard, prazepam, was added to 100 μl of plasma before a liquid–liquid extraction by 3 ml of tert-butyl methyl ether. The extracts were evaporated to dryness and reconstituted with 100 μl of mobile phase before being injected in the chromatographic system. The separation was made on a C8 column using sodium acetate buffer (pH 5):methanol:acetonitrile (35:30:35, v/v/v) as mobile phase. The detection was performed at a wavelength of 260 nm. The method was linear and has been validated over a concentration range of 2–80 mg/l. The mean precision and accuracy of the method were respectively, 10.5 and −9.1%. The mean recovery was 70.8%.
Keywords: Tipranavir; Liquid chromatography; UV detection;

Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12 ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10 ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63 ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.
Keywords: Penicillin; Liquid chromatography–tandem mass spectrometry; Residue analysis; Quantification; Plasma; Urine; Kidney;

A chromatographic immunostaining method has been developed for the determination of ginsenoside Re (G-Re) in ginseng samples on a polyethersulphone (PES) membrane. G-Re standard and the extracts of ginseng roots were applied to a PES membrane and developed by methanol–water–acetic acid (45:55:1, by volume). G-Re was clearly detected by an immunostaining method using a monoclonal antibody against G-Re. The coloring spots of G-Re were analyzed quantitatively using NIH Image software indicating at least 0.125 μg of G-Re was detectable. G-Re can be analyzed quantitatively between 0.25 and 4.0 μg.
Keywords: Ginseng; Ginsenoside Re; Chromatographic immunostaining; NIH Image software;

A method to identify and sequence recombinant mouse acetylcholinesterase (rMoAChE) including the native and organophosphate-modified active-site peptides was developed using capillary liquid chromatography with electrospray ionization, quadrupole/time-of-flight mass spectrometry. Addition of 2-propanol to the reversed-phase gradient system and a decreased gradient slope improved the peptide resolution and the signal of the active-site peptide. The highest protein coverage and active-site peptide signal were achieved when the rMoAChE:chymotrypsin ratio of 5:1 was used with digestion at 37 °C. rMoAChE and the active-site peptide were identified and sequenced from chymotryptic digests of native, methyl paraoxon-, and ethyl paraoxon-inactivated rMoAChE showing unequivocally that the exact modification site was the active-site serine.
Keywords: Acetylcholinesterase; QTOF; MS; Active site; Peptide; Chymotrypsin; Organophosphate; Methyl paraoxon; Ethyl paraoxon;

Stereoselective HPLC assay of donepezil enantiomers with UV detection and its application to pharmacokinetics in rats by Mahasen A. Radwan; Heba H. Abdine; Bushra T. Al-Quadeb; Hassan Y. Aboul-Enein; Kenichiro Nakashima (114-119).
This investigation describes a new precise, sensitive and accurate stereoselective HPLC method for the simultaneous determination of donepezil enantiomers in tablets and plasma with enough sensitivity to follow its pharmacokinetics in rats up to 12 h after single oral dosing. Enantiomeric resolution was achieved on a cellulose tris (3,5-dimethylphenyl carbamate) column known as Chiralcel OD, with UV detection at 268 nm, and the mobile phase consisted of n-hexane, isopropanol and triethylamine (87:12.9:0.1). Using the chromatographic conditions described, donepezil enantiomers were well resolved with mean retention times of 12.8 and 16.3 min, respectively. Linear response (r  > 0.994) was observed over the range of 0.05–2 μg/ml of donepezil enantiomers, with detection limit of 20 ng/ml. The mean relative standard deviation (R.S.D.%) of the results of within-day precision and accuracy of the drug were ≤10%. There was no significant difference (p  > 0.05) between inter- and intra-day studies for each enantiomers which confirmed the reproducibility of the assay method. The mean extraction efficiency was 92.6–93.2% of the enantiomers. The proposed method was found to be suitable and accurate for the quantitative determination of donepezil enantiomers in tablets. The assay method also shows good specificity to donepezil enantiomers, and it could be successfully applied to its pharmacokinetic studies and to therapeutic drug monitoring.
Keywords: Donepezil; Stereoselective; HPLC; Enantiomers; Pharmacokinetics; Chiral stationary phase;

ZT-1 is a novel acetylcholinesterase (AChE) inhibitor. It is rapidly transformed to Huperzine A (Hup A) in vitro. A simple and rapid HPLC-UV method for the simultaneous determination of ZT-1 and its metabolite Hup A in plasma is described. The chromatographic separations were achieved on a C18 ODS column (250 mm × 4.6 mm ID) using methanol-1 mmol/L ammonium acetate (70:30,v/v) as mobile phase. The flow rate was 0.7 mL/min, the detection wavelength was 313 nm and the column temperature was kept at 35 °C. Plasma samples were prepared as rapidly as possible and extracted immediately with 5 mL of chloroform:iso-propyl alcohol mixture (v/v, 9:1).The retention times of ZT-1 and Huperzine A (Hup A) were 18.7 and 14.4 min, respectively. The mean absolute recoveries of two analytes were >90%. Quantification limits were all 0.02 nmol/mL for ZT-1 and Hup A. This analytical method was reliable and convenient procedure that meets the criteria for the pharmacokinetic evaluation of ZT-1 on experimental animals.
Keywords: ZT-1; Huperzine A;

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry by Michel W.F. Nielen; Johan J.P. Lasaroms; Patrick P.J. Mulder; Johan Van Hende; J.(Hans)A. van Rhijn; Maria J. Groot (126-134).
The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17β-testosterone and 17α-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17β-testosterone and the undecylenate ester of 17β-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2–5 ng/g and 97–105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.
Keywords: Liquid chromatography; Mass spectrometry; Hair; Testosterone ester; Anabolic steroid; Boldenone;

Bioanalytical methods using liquid/liquid extraction (LLE) and liquid chromatography with electrospray tandem mass spectrometry (LC–MS/MS) are widely used. The organic extracts need to be evaporated and reconstituted, hampering further improvement of throughput and automation. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well LLE by using hydrophilic interaction chromatography with MS/MS (HILIC–MS/MS) on silica column with high organic/low aqueous mobile phase. Omeprazole, its metabolite 5-OH omeprazole, and internal standard desoxyomeprazole, were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 μl was injected onto a Betasil silica column (50 mm × 3.0 mm, 5 μm) and detected by API 3000 and 4000 with (+) ESI. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95:5:0.1 to 73.5:26.5:0.1 in 2 min). The flow rate was 1.5 ml/min with total run time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <4.4% relative standard deviation (R.S.D.) and 4.1% relative error (R.E.) for omeprazole, and 4.5% R.S.D. and 5.6% R.E. for 5-OH omeprazole, respectively.
Keywords: Omeprazole; 5-OH omeprazole; HILIC–MS/MS; Liquid/liquid extraction;

Ezetimibe (Ezetrol®) is a novel cholesterol lowering drug which disposition is not fully understood in man. We developed a selective and high-sensitive assay to measure serum concentration–time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies. Ezetimibe glucuronide, the major metabolite of ezetimibe was determined by enzymatic degradation to the parent compound. Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-hydroxychalcone as internal standard and liquid chromatography coupled via an APCI interface with tandem mass spectrometry (LC–MS/MS) for detection. The chromatography (column XTerra® MS, C18, 2.1 mm × 100 mm, particle size 3.5 μm) was done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 μl/min). The MS/MS analysis was performed in the negative ion mode (m/z transition: ezetimibe 408–271, internal standard 223–117). The validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001–0.015 μg/ml and 0.001–0.2 μg/ml; urine and fecal homogenate 0.025–10 μg/ml and 0.1–20 μg/ml, respectively. The assay was successfully applied to measure ezetimibe disposition in two subjects genotyped for the hepatic uptake transporter SLCO1B1.
Keywords: Ezetimibe; Ezetimibe glucuronide; Bioanalytics; LC–MS/MS; Human serum; Urine; Feces; OATP-C; SLCO1B1;

Quantification of ACE inhibiting peptides in human plasma using high performance liquid chromatography–mass spectrometry by Chris J. van Platerink; Hans-Gerd M. Janssen; Roos Horsten; Johan Haverkamp (151-157).
An HPLC-MRM–MS method was developed for the quantification of 17 small ACE inhibiting (ACEI) peptides in plasma samples collected from human volunteers after the consumption of a peptide-enriched drink. The assay shows the high selectivity and sensitivity necessary to monitor small changes in the levels of the ACEI peptides after consumption of drinks developed to effect lowering of the blood pressure. Four different sample preparation methods were tested and evaluated. The final sample preparation method selected is simple and effective and consists mainly of the removal of proteins by acidification and heating, followed by a large volume injection. Additional sample preparation steps such as solid phase extraction and liquid/liquid partitioning were studied. Although they resulted in cleaner extracts, losses of specific peptides such as SAP were frequently seen. The isotope labeled form of one of the peptides to be quantified, [U13C]IPP, was used as an internal standard. The limit of detection of the assay is below 0.01 ng ml−1. The limit of quantification is between 0.05 and 0.2 ng ml−1, which is approximately 10% of the expected peptide concentration in plasma based on a normal diet. The intra- and inter-day relative standard deviations for all peptides have shown to be below 25% and the method has an accuracy of better than 75%. The long-term stability is good. At least 200 samples could be analysed before the system had to be cleaned. The assay has been successfully applied to blood samples collected from volunteers during a human trial.
Keywords: Bioactive peptides; LC–MS; Plasma; Angiotensin-I-converting enzyme inhibitors;

Four sample preparation methods, (1) solvent (SOL), (2) saponification and solvent (SP), (3) ultrasound assisted solvent (UA), and (4) saponification and ultrasound assisted solvent (SP-UA), were used for quantifying lutein in chicken liver samples by HPLC. The lutein concentrations obtained by using SOL, UA, SP, and SP-UA were significantly different with values from 10.4 μg/g (UA) to undetected (SOL). Efficiency of the four different methods for extracting lutein from high to low were the UA, SP, SP-UA, and SOL method. The measured value of lutein in the liver sample using the UA method was approximately two and three times higher than that obtained from the SP and SP-UA method, respectively. The methods with saponification significantly affected the stabilities of lutein in liver samples. The lutein concentration measured with the solvent only method was either much lower than any of the other extraction methods or undetectable. This indicated that little lutein in those samples was in a form that could be extracted directly by solvent. Compared with the saponification method, the ultrasound assisted solvent method could effectively extract lutein from sample matrix and thus avoid chemical degradation reactions, which would be especially important for complex biological tissue such as liver.
Keywords: Lutein; Ultrasound; Saponification; HPLC; Carotenoid; Extraction; Chicken liver;

Histamine content in fish may increase by decarboxylation of free histidine to values that can be toxic, if storage conditions are not well controlled. We have studied the influence of storage temperature and time of freezing on histamine formation in the anchovy, Engraulis encrasicholus (L., 1758), for which little information is available. Analysis, carried out by capillary zone electrophoresis (CZE) without sample pre-treatment, was very simple, fast and reproducible. Results indicate that temperatures above 20 °C notably increase histamine production, whereas freezing can clearly prevent or slow down the process.
Keywords: Histamine; Anchovy; Capillary zone electrophoresis; Scombroid fish; Clupeid fish;

A liquid chromatographic–mass spectrometric evidence of dihydrosanguinarine as a first metabolite of sanguinarine transformation in rat by Jitka Psotová; Bořivoj Klejdus; Rostislav Večeřa; Pavel Kosina; Vlastimil Kubáň; Jaroslav Vičar; Vilím Šimánek; Jitka Ulrichová (165-172).
Adult rats were orally administered with a single dose of sanguinarine (10 mg SA per 1 kg body weight) in 1.0 ml water. In the plasma and the liver, dihydrosanguinarine (DHSA) was identified as a SA metabolite by high performance liquid chromatography–electrospray ionization mass spectrometry (HPLC/ESI-MS). Significantly higher levels of DHSA were found in both the plasma and the liver in comparison with those of SA. SA and DHSA were not detected in the urine. The formation of DHSA might be the first step of SA detoxification in the organism and its subsequent elimination in phase II reactions. Benz[c]acridine (BCA), in the literature cited SA metabolite, was found neither in urine nor in plasma and liver.
Keywords: Quaternary benzo[c]phenanthridine alkaloid; Sanguinarine; Rat; Metabolite; Liquid chromatography–mass spectrometry; Liver; Plasma; Dihydrosanguinarine; Benz[c]acridine;

Threonine was oxidized into acetaldehyde at 0 °C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 μl sample solution and the determination limit of threonine was 1 nmol in 200 μl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.
Keywords: Threonine; Gas chromatography; Determination;

Gas chromatography/mass spectrometry characterization of urinary metabolites of danazol after oral administration in human by Rodny Montes de Oca Porto; Ariana Rodríguez Fernández; Dayamín Martínez Brito; Teresa Correa Vidal; Ahiram Lopez Diaz (178-183).
Danazol (17α-pregna-2,4-dien-20-yno [2,3-d]-isoxazol-17β-ol), is a synthetic derivative of ethisterone, structurally related to stanozolol. For this reason its use as doping agent has been investigated. Danazol (Runch®) (200 mg) were orally administered to two healthy male volunteers. Urine samples were colleted up to 1-week post-dose. Four new metabolites have been identified in addition to the five previously reported. We propose the monitorization of 6β-hydroxy-2-hydroxymethyl-1,2-dehydroethisterone and 6β,16ξ-dihydroxy-2ξ-hydroxymethyl-ethisterone by free fraction analysis. In a same way, we proposed to detect the principal isomer of a mono-hydroxylated metabolite of 6β-hydroxy-2ξ-hydroxymethylethisterone in the conjugated fraction. We conclude that new metabolites can be included for the detection of danazol abuse since the main metabolite ethisterone is excreted relatively fast in urine.
Keywords: Danazol; Ethisterone; Doping control;